Nucleoside-Modified mRNA Vaccines Protect IFNAR-/- Mice against Crimean-Congo Hemorrhagic Fever Virus Infection

Abstract

Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is on the World Health Organizations' list of prioritized diseases and pathogens. With global distribution, high fatality rate, and no approved vaccine or effective treatment, CCHF constitutes a threat against global health. In the current study, we demonstrate that vaccination with nucleoside-modified mRNA-lipid nanoparticles (mRNA-LNP), encoding for the CCHFV nucleoprotein (N) or glycoproteins (GcGn) protect IFNAR^-/- mice against lethal CCHFV infection. In addition, we found that both mRNA-LNP induced strong humoral and cellular immune responses in IFNAR^-/- and immunocompetent mice and that neutralizing antibodies are not necessary for protection. When evaluating immune responses induced by immunization including CCHFV Gc and Gn antigens, we found the Gc protein to be more immunogenic compared with the Gn protein. Hepatic injury is prevalent in CCHF and contributes to the severity and mortality of the disease in humans. Thus, to understand the immune response in the liver after infection and the potential effect of the vaccine, we performed a proteomic analysis on liver samples from vaccinated and control mice after CCHFV infection. Similar to observations in humans, vaccination affected the metabolic pathways. In conclusion, this study shows that a CCHFV mRNA-LNP vaccine, based on viral nucleo- or glycoproteins, mediate protection against CCHFV induced disease. Consequently, genetic immunization is an attractive approach to prevent disease caused by CCHFV and we believe we have necessary evidence to bring this vaccine platform to the next step in the development of a vaccine against CCHFV infection. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a zoonotic pathogen causing Crimean-Congo hemorrhagic fever (CCHF), a severe fever disease. CCHFV has a wide distribution and is endemic in several areas around the world. Cases of CCHF are also being reported in new areas, indicating an expansion of the disease, which is of high concern. Dispersion of the disease, high fatality rate, and no approved vaccine makes CCHF a threat to global health. The development of a vaccine is thus of great importance. Here we show 100% protection against lethal CCHFV infection in mice immunized with mRNA-LNP encoding for different CCHFV proteins. The vaccination showed both robust humoral and cellular immunity. mRNA-LNP vaccines combine the ability to induce an effective immune response, the safety of a transient carrier, and the flexibility of genetic vaccines. This and our results from the current study support the development of a mRNA-LNP based vaccine against CCHFV.

Keywords: Crimean-Congo hemorrhagic fever virus; Gc; Gn; IFNAR mice; N; T-cell immunity; mRNA vaccine; neutralizing antibodies.

FIG 1

Survival and viral RNA in mRNA-LNP CCHFV-infected IFNAR^−/− mice. Schematic drawing illustrating the immunization schedule for (a) A129 IFNAR^−/− mice and (b) immunocompetent 129S2 mice. Each mouse received two immunizations with 3 weeks between. The mice received 10 μg of each specified mRNA-LNP administered through intradermal injections. Five weeks post last immunization, all immunocompetent mice were euthanized, while the IFNAR^−/− mice were challenged with CCHFV IbAr10200 via intraperitoneal injection and followed for 2 weeks. (c) Kaphlan-Meire graph showing survival of vaccinated and control IFNAR^−/− mice after CCHFV challenge (GcGn, N, GcGn+N, and control). At the time of euthanasia, viral RNA in (d) serum, (e) spleen, and (f) liver was measured using qRT-PCR. For serum, data is shown as mean CT values ± geometric standard deviation. Dashed line indicate limit of detection. For spleen and liver, the graphs display fold change ± geometric standard deviation relative to the control group. ****P < 0.0001. P values calculated with one-way ANOVA with Dunnett’s multiple comparison test.

FIG 2

Antibody response in IFNAR^−/− mice due to mRNA-LNP vaccination. (a) Antibody titer in pooled, diluted serum from IFNAR^−/− mice immunized with N or GcGn+N mRNA-LNP after the first (T1) and second (T2) immunization measured with VectorBest ELISA. (b) Titers of antibodies against CCHFV Gc or (c) Gn in pooled, diluted serum from IFNAR^−/− mice immunized with GcGn mRNA-LNP measured with in-house ELISAs coated with CCHFV Gc or Gn protein, respectively. Data is shown as mean ± standard deviation of two technical replicates. Dotted lines indicate limit of detection. (d) Pooled, diluted serum from IFNAR^−/− mice for each vaccine group after two immunization (T2) were evaluated for neutralization capacity. Data is shown as percent inhibition compared with control animals for each dilution. T1 is represented by white symbols: N, GcGn+N, GcGn, control. T2 is represented by black symbols: N, GcGn+N, GcGn, and control. OD, optical density.

FIG 3

Antibody response in immunocompetent mice due to mRNA-LNP vaccination. (a) Antibody titer in pooled, diluted serum from immunocompetent mice immunized with N or GcGn+N mRNA-LNP after the first (T1) and second (T2) immunization measured with VectorBest ELISA. (b) Titers of antibodies against CCHFV Gc or (c) Gn in pooled, diluted serum from immunocompetent mice immunized with GcGn mRNA-LNP measured with in-house ELISAs coated with CCHFV Gc or Gn protein, respectively. Data is show as mean ± standard deviation of twp technical replicates. Dotted lines indicate limit of detection. (d) Pooled, diluted serum from immunocompetent mice for each vaccine group after two immunization (T2) were evaluated for neutralization capacity. Data is shown as percent inhibition compared with control animals for each dilution. T1 is represented by white symbols: N, GcGn+N, GcGn, control. T2 is represented by black symbols: N, GcGn+N, GcGn, and control. OD, optical density.

FIG 4

mRNA-LNP immunization induces CCHFV-specific cellular response. Fresh splenocytes from the immunocompetent mice after two immunizations were stimulated with pooled, overlapping peptides based on the CCHFV Gc, Gn, or N protein. ELISpot was used to determine the number of IFN-γ SFCs per 10⁶ splenocytes in (a) GcGn,(b) N, (c) GcGn+N, and (d) control vaccinated mice. Each peptide pool for N and Gc contained eight peptides, while Gn-peptide pools contained seven peptides. Each pool was performed in triplicate and data is shown as geometric mean ± geometric standard deviation. As control antigens OVA-CTL, OVA-Th, ConA, and medium alone were used and cut-off was set at 50 SFCs/10⁶ splenocytes. Amino acid position (aa#) is based on CCHFV nucleoprotein, NCBI accession number NP_950237 and CCHFV glycoprotein precursor, NCBI accession number NP_950235.

FIG 5

Proteomic analysis of GcGn-vaccinated versus control mice. (a) PCA trajectory score plots labeled for GcGn-vaccinated and control mice. (b) Volcano plots of proteins with differential abundance between GcGn-vaccinated and control mice. Upregulated proteins are represented in red, while proteins downregulated are represented in green. FDR < 0.05. (c) Cytoscape network of KEGG pathways enriched in proteins differing between GcGn-vaccinated and control mice. Nonsignificant pathways are gray. Significant upregulated pathways are represented in red and downregulated in green. Node size is proportional to the number of proteins associated with a pathway. Edge width is proportional to the number of proteins shared between two pathways. (d) Bubble plot representing significant pathways from the KEGG pathway map “Metabolism” enriched in proteins with significant higher abundance in GcGn vaccinated mice compared with control. Pathways are ordered by adjusted P value and size is proportional to the number of proteins.

FIG 6

Proteomics analysis of N-vaccinated versus control mice. (a) PCA trajectory score plots labeled for N-vaccinated and control mice. (b) Volcano plots of proteins with differential abundance between N-vaccinated and control mice. (c) Cytoscape network of KEGG pathways enriched in proteins differing between control and GcGn. (d) Bubble plot representing KEGG pathways from the “Metabolism” pathway map enriched in the protein with higher intensity in N-vaccinated mice compared with control. (e) Venn diagram of protein with differential abundance between control, GcGn-, and N-vaccinated mice. (f) Barplot of the top 10 biological KEGG pathways enriched in proteins differing between control and, respectively, GcGn- and N-vaccinated groups of mice. Pathways were ordered by adjusted P value. Upregulated pathways are represented in red and downregulated pathways in green. Number of proteins associated with each pathway is indicated on the bar.